Inhibitory activity against carbonic anhydrase IX and XII as a candidate selection criterion in the development of new anticancer agents

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Inhibitory activity against carbonic anhydrase IX

and XII as a candidate selection criterion in the

development of new anticancer agents

Mikhail Krasavin , Stanislav Kalinin , Tatiana Sharonova & Claudiu T. Supuran

To cite this article:

Mikhail Krasavin , Stanislav Kalinin , Tatiana Sharonova & Claudiu T. Supuran

(2020) Inhibitory activity against carbonic anhydrase IX and XII as a candidate selection criterion in

the development of new anticancer agents, Journal of Enzyme Inhibition and Medicinal Chemistry,

35:1, 1555-1561, DOI: 10.1080/14756366.2020.1801674

To link to this article: https://doi.org/10.1080/14756366.2020.1801674

© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

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SHORT COMMUNICATION

Inhibitory activity against carbonic anhydrase IX and XII as a candidate selection

criterion in the development of new anticancer agents

Mikhail Krasavin

, Stanislav Kalinin

, Tatiana Sharonova

and Claudiu T. Supuran

a

Institute of Chemistry, St. Petersburg State University, St. Petersburg, Russia;bNeurofarba Department, Section of Pharmaceutical Sciences, University of Florence, Florence, Italy

ABSTRACT

Analysis of the literature data reveals that while inhibition of cancer-related carbonic anhydrase IX and XII isoforms continues to be an important enrichment factor for designing anticancer agent development libraries, exclusive reliance on the in vitro inhibition of these two recombinant isozymes in nominating candidate compounds for evaluation of their effects on cancer cells may lead not only to identifying numerous compounds devoid of the desired cellular efficacy but also to overlooking many promising can-didates which may not display the best potency in biochemical inhibition assay. However, SLC-0111, now in phase Ib/II clinical trials, was developed based on the excellent agreement between thein vitro, in vivo and more recently, in-patient data.

ARTICLE HISTORY Received 4 July 2020 Accepted 21 July 2020 KEYWORDS Carbonic anhydrase inhibitors; cancer-related IX and XII isoforms; anticancer agents; screening funnel; enrichment factor

Introduction

Tumour growth and proliferation are strongly associated with hyp-oxic stress due to poor vascularisation and oxygen deprivation of neoplastic tissues1_{. Adaptive metabolic changes observed in} can-cer cells include elevated production of acidic metabolites which leads to tumour acidosis2–4. Human carbonic anhydrase (hCA) iso-forms IX and XII are crucial effectors that regulate extracellular pH thus mediating cancer cell proliferation, invasion, and metasta-sis5,6. These membrane-bound proteins are zinc metalloenzymes that catalyse the reversible hydration of CO2to bicarbonate anion and proton on cell surface (Equation (1))7_{. Many studies confirmed} hCA IX/XII upregulation in hypoxic tumours8,9

. In particular, hCA IX which has limited expression in normal tissues, is a marker of aggressive and drug-resistant cancer cell phenotypes indicating poor prognosis for patients10. Arguably, hCA XII is widely distrib-uted in the human body and considered a marker for less malig-nant tumours11. Targeting the catalytic activity of the cell surface carbonic anhydrases has been and continues to be considered a promising therapeutic approach in the antineoplastic field, either for suppressing tumour growth or overcoming drug resistance of cancer cells12,13.

CO2þ H2OÀHCO3 þ Hþ (1)

Much progress has been made in the last decade towards anti-cancer agents based on small-molecule inhibitors of hCA IX and/ or XII14,15_{. These efforts typically involved screening of compound} libraries in search for potent and selective blockers of these iso-zymes, followed by detailedin vitro and in vivo characterisation of the most promising candidates. Many new compounds have been thus identified capable of inhibiting recombinant hCA IX and/or XII in the low nanomolar to subnanomolar range with remarkable

selectivity over other carbonic anhydrase isoforms. These chemo-types often contained a primary sulphonamide or sulfamate (e.g. 1 and 2) group or were based on a coumarin and sulfocoumarin core (such as 3 and 4, respectively)16–19. Of these classes, ureido-substituted benzene sulphonamides (USBs) made the most pro-gress with SLC-0111 recently entering phase Ib/II clinical trials (Figure 1)20,21_.

Despite significant advances, the discovery of hCA-targeted anticancer agents is not a straightforward endeavour, with many aspects remain unclear. In fact, our own efforts as well as those reported in the literature often yield structures, which display frus-tratingly modest antiproliferative effect, although possessing pro-found inhibition of isolated hCA IX/XII. Such inconsistencies occurring between the ability of somehCA inhibitors to block the recombinant enzyme catalytic function and their effects on cancer cells constitute a challenge that has been much less addressed, in comparison to those related to the SAR and selectivity stud-ies22–24. Thus, although the potential of hCA IX and XII as anti-cancer targets is supported by much evidence, including the well-documented efficacy of hCA-inhibitory antibodies in vitro and in vivo, the success rate of small-molecule inhibitors upon the transition to cell culture setting is still below the desirable level18,25–29. Taking this into account, attempts to re-evaluate the conventional drug discovery workflow that has existed in this field are of importance. Indeed, while factors affecting the activity in cells are poorly understood, the drug-discovery funnel beginning with recombinant protein-based profiling should be critically eval-uated. Therefore, we undertook a literature survey and analysis regarding possible disconnects for hCA inhibitors, leading to a dramatic change of the compound’s activity upon the transition from the recombinant isolated protein to the cell-based models.

CONTACT Mikhail Krasavin m.krasavin@spbu.ru Institute of Chemistry, St. Petersburg State University, St. Petersburg, Russia; Claudiu T. Supuran

claudiu.supuran@unifi.it Neurofarba Department, Section of Pharmaceutical Sciences, University of Florence, Florence, Italy

Supplemental data for this article is available online athere.

ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY 2020, VOL. 35, NO. 1, 1555_–1561

Discussion

Disappointingly, a large fraction of publications on the subject reported compounds’ cytotoxicity data obtained in normoxic con-ditions thus making the involvement of hCA IX and XII debate-able30–45. Although such testing can be relevant in some cases, we consider these conditions irrelevant as long as antiproliferative activity ofhCA inhibitors is concerned. This is primarily due to lim-ited expression of the target isoforms, if any, reported for many cancer cell lines under these conditions8,46. The studies which employ normoxic MCF-10A cells lackinghCAIX/XII on their surface are illustrative47. Furthermore, some investigations involved cell lines displaying robust hCA IX activity in the cytosol, such as MDA-MB-231 and MCF-748. The said non-default localisation of hCA IX could lead to overestimation of the protein expression and activity on the cell membrane, thus leading to the problematic interpretation of the data obtained16. Thus, the choice of experi-mental conditions often renders the results ambiguous, making them rather difficult to analyse.

To our delight, however, there is a large number of mechanis-tically relevant experiments reported in the literature which address the ability ofhCA IX/XII inhibitors to block the growth of hypoxic cancer cells overexpressing the target isozymes49–67. Moreover, a certain cohort of studies revealed compounds possessing both favourable hCA inhibitory profile and significant anticancer activity, as exemplified by structures 5–13 (Figure 2)49–56,58–61,64–67. In fact, quite a number of single-digit nanomolarhCA IX/XII inhibitors significantly suppressed cancer cell growth. Additional data underscoring the specificity of this antiproli-ferative action in cancer over normoxic and non-cancerous cells highlight the potential of these findings for practical applications. Meanwhile, the drug-like character of some frontrunners makes them intriguing starting points for medicinal chemistry optimisation. Once selected based on their hCA inhibitory profiles, these com-pounds might be useful indeed in designing antineoplastic drugs either of combinative or single-agent use49–53,55,56,61,65.

In contrast to the aforementioned examples of good correl-ation between hCA IX/XII inhibition and cytotoxicity against can-cer cells in hypoxic conditions, the reverse is true for a strikingly large number of examples50,55–57,60–63,67. In these cases, many potenthCA IX/XII inhibitors, such as 14–19, did not possess a sub-stantial activity against cancer cells. Furthermore, these examples often share structural similarity with the hit compounds originat-ing from the same screenoriginat-ing series (cf. examples in Figure 2). Despite the obvious resemblance, no effect observed under identi-cal conditions, again, indicates our limited understating of the fac-tors influencing hCA inhibitors activity in the intact cells (Figure 3)50,55,56,61–63. According to the existing discovery work-flow in this field, if these compounds are selected for the detailed evaluation based solely on their enzyme inhibitory profile, this could have diverted the investigator’s attention from potentially efficacious anticancer agents.

A large portion of compounds may be overlooked in the anti-cancer agent development projects because of their moderate inhibitory properties towards hCA IX/XII cancer-related isozyme duo. This fairly populated category of compounds possessing moderate to low inhibitory properties towards recombinant hCA IX/XII features many compounds that are based on privileged che-motypes (e.g. 20–25). Evidently, in most studies, these relatively inactive molecules would have not been progressed based solely on their inhibitory profile. At the same time, several reports reveal that and unexpectedly good cytotoxicity towards cancer cells can be demonstrated by such compounds (Figure 4)30,33,34,68–70.

These occasional findings supported the questioning of the dir-ect predictive power of hCA IX/XII inhibitory profile for a com-pound’s anticancer activity. Understanding the significance of the candidate molecule prioritisation problem in question is essential while ignoring it may lead to many promising compounds being overlooked in want of more potent hCA IX/XII inhibitors. Aiming to draw a more informative picture, we have graphically repre-sented the relationship between the anticancer effect (in this case,

Figure 1. Potent and selective hCA IX/XII inhibitors from among sulphonamides (1), sulfamates (2), coumarins (3), and sulfocumarins (4) and SLC-0111 with its CA inhibition profile.

irrespective of the specific experimental conditions employed in various studies) and the hCA IX/XII inhibitory activity of the com-pounds30–45,49–58,71–74. In order to summarise the somewhat het-erogeneous literature data, we have visualised the reported antiproliferative agents in the plots with theY-axis displaying Ki’s against hCA IX (Figures S1 and S2) and hCA XII (Figures S3 and S4). Several groups of compounds were distinguished and marked by a different colour with regard to their effect on cancer cells: (1) <50% cell survival at 10 mM (or IC50¼0.1–10 mM), green; (2) <50% cell survival at 50 mM (or IC50¼11–50 mM), blue; (3) IC50 51–100 mM, tangerine; (4) IC50¼101–150 mM, cyan; (5) IC50¼151–200 mM, magenta; (6) IC50 >200 mM, red; (7) <50% cell growth inhibition at 10 or 50 mM, yellow. As is evident from

Figures S1–S4, potent cancer cell growth suppressors can possess vastly different levels of inhibitory activity against hCA IX/XII. However, the low nanomolar region of Ki values appears to be most populated with compound possessing strong effect on can-cer cells (Figures S1 and S3). Meanwhile, a fairly large portion of compounds did not possess noticeable antiproliferative activity while displaying various levels of Ki values towards recombinant hCAs (Figures S2 and S4). The prevailing occurrence of these com-pounds in the nanomolar range ofhCA IX/XII inhibitory activity is

likely the result of the currently applied drug discovery funnel where the besthCA IX/XII inhibitors are primarily selected for sub-sequent evaluation for their effects on cancer cells.

The supremacy of in vitro potency for drug discovery decision making has been much debated with respect to different fields of medicinal chemistry75. In complex biological environment, mul-tiple variables such as ligand residence time or metabolic stabil-ity76,77 can determine the observed efficacy. However, the said parameters are not routinely looked at when screening for poten-tialhCA IX/XII inhibitors. While tens to hundreds nanomolar levels of on-target activity prevail among clinically used drugs77, such activity levels are easily achievable for primary sulphonamides hCA IX and\or XII isoforms, as can be deduced from Figures S1–S4. This gives us an idea of a different approach to the discov-ery of anticancer agents based on hCA IX/XII inhibition. Considering, that multiple factors appear to be at play when cyto-toxicity towards cancer cells is concerned, front-loadinghCA IX/XII inhibition as a candidate selection criterion should be taken with caution. In vitro screening is indispensable for identifying chemo-types with a clear tendency to inhibit the target isozymes. Once identified and expanded into larger, SAR-informative follow-on libraries, these candidate chemotypes should perhaps be screened

Figure 2. PotenthCA IX/XII inhibitors displaying good translation of their activity into antiproliferative activity against cancer cells under hypoxia.

Figure 3. PotenthCA IX/XII exhibiting poor antiproliferative activity under hypoxic conditions.

Figure 4. Sulphonamides and coumarins demonstrating potent antiproliferative properties despite being weak inhibitors of the recombinanthCA IX/XII. 1558 M. KRASAVIN ET AL.

primarily against cancer cells so as not to overlook promising can-didates which may not necessarily be endowed with the besthCA IX/XII inhibitory potency.

Conclusions

Literature data indicate that inhibition of the recombinant protein possesses a somewhat limited predictive power for the com-pounds’ ability to block cancer cell growth. Not questioning the role of the cell surface hCA isoforms in tumour growth and sur-vival, this conclusion, however, highlights the imperfection of the existing approach to the design and discovery of antineoplastic drugs in this field. In fact, by selecting compounds forin vitro and in vivo characterisation based solely on their inhibitory profile, one could discard many promising hit-structures. Resources expended for optimising molecules’ potency and selectivity towards the tar-get recombinant protein may also be wasted as they do not necessarily ensure antiproliferative activity. On the other hand, a wealth of positive examples in the literature of a strong correl-ation between hCA IX/XII inhibition and cytotoxicity towards can-cer cells indicates that demonstrated tendency of new chemotypes to inhibit these isoforms can serve as a robust enrich-ment factor for designing screening libraries that target cancer cells. Moreover, detailed SAR studies of different carbonic anhy-drase inhibitors with regard to hCA IX and/or XII are of import-ance due to the significimport-ance of developing new ligands of these targets for tumour imaging, drug delivery and other purposes78_.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Funding

This research was supported by the Russian Federation Government Megagrant 14.W03.031.0025. Tatiana Sharonova is grateful to the Russian Foundation for Basic Research for the graduate student research grant (project 19-33-90017).

ORCID

Claudiu T. Supuran http://orcid.org/0000-0003-4262-0323

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